Inkjet printing of tissues and cells

ABSTRACT

Provided herein is an apparatus for printing cells which includes an electrospinning device and an inkjet printing device operatively associated therewith. Methods of making a biodegradable scaffold having cells seeded therein are also provided. Methods of forming microparticles containing one or more cells encapsulated by a substrate are also provided, as are methods of forming an array of said microparticles.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application Ser. No. 61/028,761, filed Feb. 14, 2008,the disclosure of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention concerns inkjet printing of viable cells andarrays of cells so produced.

BACKGROUND OF THE INVENTION

In the interdisciplinary field of tissue engineering, powerful newtherapies are being developed to address structural and functionaldisorders of human health by utilizing living cells as engineeringmaterials. In some areas of tissue engineering, researchers are creatingtwo- and three-dimensional tissues and organs from combinations of cellsin order to repair or replace diseased or damaged tissues.

Organ printing using inkjet printing is evolving to become moreoptimized by delivering multiple cell types and scaffolds to targetspecific regions. However, most current printing technologies arelimited to hydrogel as the primary scaffold for tissue constructs. Amajor disadvantage of hydrogels is their low mechanical strength, whichmakes handling and in vivo application difficult, particularly forload-bearing implants. Alternative methods to create implants havingenhanced mechanical properties are needed.

Inkjet printing technology is based on the rapid creation and release ofliquid droplets, followed by their precise deposition on a substrate.Recently, this technology has generated increased interest in biomedicalmicro-fabrication, as it offers a practical and efficient method todispense biological and/or material elements, including living cells(Boland et al., 2007, “Drop-on-demand printing of cells and materialsfor designer tissue constructs,” Materials Science & EngineeringC-Biomimetic and Supramolecular Systems, 27(3), pp. 372-376; Xu et al.,2006, “Viability and electrophysiology of neural cell structuresgenerated by the inkjet printing method,” Biomaterials, 27(19), pp.3580-3588; Xu et al., 2005, “Inkjet printing of viable mammalian cells,”Biomaterials, 26(1), pp. 93-99; Xu et al., 2004, “Construction ofhigh-density bacterial colony arrays and patterns by the ink-jetmethod,” Biotechnol Bioeng, 85(1), pp. 29-33).

The cell represents the basic unit of life and as such, it has becomethe focus of extensive research. Single-cell analysis is advantageousover conventional bulk cell methods as it allows complex andheterogeneous biological systems to be monitored at their most basiclevel (Shoemaker et al., 2007, “Multiple sampling in single-cell enzymeassays using capillary electrophoresis with laser-induced fluorescencedetection,” Anal Bioanal Chem, 387(1), pp. 13-15). In recent years,single-cell based analytical devices have been increasingly applied in awide range of biomedical applications, such as single-cell assays (Lu etal., 2004, “Recent developments in single-cell analysis,” AnalyticaChimica Acta, 510(2), pp. 127-138), high throughput screening (Anderssonet al., 2004, “Microtechnologies and nanotechnologies for single-cellanalysis,” Curr Opin Biotechnol, 15(1), pp. 44-49; Brehm-Stecher et al.,2004, “Single-cell microbiology: tools, technologies, and applications,”Microbiol Mol Biol Rev, 68(3), pp. 538-559), single-cell proteinlibraries and gene expression (Fukuda et al., 2006, “Construction of acultivation system of a yeast single cell in a cell chip microchamber,”Biotechnology Progress, 22(4), pp. 944-948; Janicki et al., 2004, “Fromsilencing to gene expression: Real-time analysis in single cells,” Cell,116(5), pp. 683-698), and miniature biosensors (Maruyama et al., 2005,“Immobilization of individual cells by local photo-polymerization on achip,” Analyst, 130(3), pp. 304-310). These devices usually require theuse of appropriate carriers to deliver and manipulate single cells.

Recently, microparticles that contain individual living cells have beenapplied in single-cell analytical systems as effective carriers (He etal., 2005, “Selective encapsulation of single cells and subcellularorganelles into picoliter- and femtoliter volume droplets,” Anal Chem,77(6), pp. 1539-1544). These particles provide easy handling of singlecells and enhance detection efficiency (Huebner et al., 2007,“Quantitative detection of protein expression in single cells usingdroplet microfluidics,” Chemical Communications (12), pp. 1218-1220).Moreover, these particles have been used as micro-reactors to enhanceand accelerate chemical and biochemical screening (Song et al., 2006,“Reactions in droplets in microfluidic channels,” AngewandteChemie-International Edition, 45(44), pp. 7336-7356). This providessingle cell analytical devices with new capabilities and improveddetection efficiency (Taly et al., 2007, “Droplets as Microreactors forHigh-Throughput Biology,” Chembiochem, 8(3), pp. 263-272).

Currently, single-cell microparticles are mainly fabricated usingmicrofluidic based methods (He et al., 2005, “Selective encapsulation ofsingle cells and subcellular organelles into picoliter- and femtolitervolume droplets,” Anal Chem, 77(6), pp. 1539-1544; Huebner et al., 2007,“Quantitative detection of protein expression in single cells usingdroplet microfluidics,” Chemical Communications (12), pp. 1218-1220;Song et al., 2006, “Reactions in droplets in microfluidic channels,”Angewandte Chemie-International Edition, 45(44), pp. 7336-7356).However, these methods have some limitations. For example, micro-fluidicapproaches are usually limited to specific geometries because theyrequire laminar fluid flow to produce microparticles (Khademhosseini etal., 2004, “Layer-by-layer deposition of hyaluronic acid andpoly-L-lysine for patterned cell cocultures,” Biomaterials, 25(17), pp.3583-3592). These methods can only create small quantities of particles,since there are a limited number of micro-channels within these devices.Furthermore, the costly equipment, specialized material, and extensiveexpertise required for operation of these devices may further limit theuse of these methods in single cell particle fabrication.

Thus, there is an acute need for more efficient approaches that canrapidly generate single cell microparticles with ease.

SUMMARY OF THE INVENTION

Provided herein is an apparatus for printing cells, including: (a) anelectrospinning device having a high voltage power supply; and/or (b) aninkjet printing device operatively associated with the electrospinningdevice. In some embodiments, the apparatus includes a three dimensionalplotter operatively connected with the electrospinning device and/orinkjet printing device. In some embodiments, the apparatus includes acontroller operatively connected to the electrospinning device and/orinkjet printing device. In some embodiments, the high voltage supply isconductively isolated from, e.g., the controller and/or plotter (e.g.,by optics or wireless communication).

Methods of making a biodegradable scaffold having cells seeded thereinare also provided, including one or more of the steps of: (a) forming abiodegradable substrate by electrospinning; and then (b) printing viablecells on said substrate. In some embodiments, the forming step and theprinting step are each performed two or more times in sequence to make abiodegradable scaffold having multiple layers.

Methods of treating a subject in need thereof are provided, includingthe step of implanting a biodegradable scaffold as described herein.

Methods of forming microparticles comprising, consisting of orconsisting essentially of one or more cells encapsulated by a substrateare provided, including the step of printing a composition comprisingthe cells and the substrate (e.g., alginate).

Also provided are methods of forming an array of microparticlescomprising one or more cells encapsulated by a substrate, including oneor more of the steps of: providing an inkjet printing device, saiddevice comprising at least one inkjet printer cartridge; loading acomposition comprising said cells and said substrate into said printercartridge; and printing said composition in an organized pattern.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationset forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of dual inkjet and electrospinning apparatusused in Example 1. Polymer is loaded into the electrospinning syringepump (A) with a ground charge attached to the polymer pump. The lowerportion of the petri dish was charged with −20 kV. Chondrocytessuspended within a fibrin and collagen matrix were plated using aninkjet valve (B).

FIG. 2. Schematic diagram of layer-by-layer arrangement of PCL andchondrocytes created by electrospinning and inkjet printing in series.

FIG. 3. Photograph of PCL-chondrocyte tissue construct formed bylayer-by-layer arrangement.

FIG. 4. SEM microscopy images of layered cartilage constructs, showingthe inkjet printed layer situated between two electrospun layers (toppanel a, 500×), and higher magnification of the electrospun PCL layer(bottom left panel b, 4000×) and inkjet printed chondrocyte, collagenand fibrin layer (bottom right panel c, 4000×).

FIG. 5. An image of the inkjet printed layer reveals that printedchondrocytes attached onto the collagen/fibrin layer and produce elasticcartilage.

FIG. 6. Testing of mechanical properties revealed that hybrid constructsshowed higher Young's modulus and UTS than printed alginate scaffolds.

FIG. 7. Histology showed the production of cartilage-specificextracellular matrix (ECM) by the presence of cells, GAGs, and Type II &IV collagen within the printed constructs.

FIG. 8. In vivo magnetic resonance imaging (MRI) showed cartilage takingshape after 2-week implantation into nude mice.

FIG. 9. Hybrid constructs produced cartilage-specific matrix in vivo.Masson trichrome and safranin O staining of printed constructs 8 wkspost-implantation.

FIG. 10. Schematic diagram of printing apparatus according to someembodiments.

FIG. 11. Phase contrast images of printed beta-TC6 cell microparticles.(A): Many individual particles were dispersed in medium 1 day afterculture. Most particles contained single or just a few cells, and veryfew particles were without cells. (B): High magnification of amicroparticle containing a single cell 1 day after culture. (C):Individual microparticle contained beta-TC6 cells 1 month after culture.The microparticle maintained its original structure and the cells werestill entrapped inside the particle. Magnifications are as follows:100×(A) and 400×(B and C).

FIG. 12. The number of entrapped cells with respect to varying cellconcentrations. As the cell concentration in the print solutionincreased, the number of printed particles containing cells increased(shown in gray bars). However, the number of microparticles containingonly a single cell decreased significantly with increase in cellconcentration (shown in black bars). (*P<0.01, n=10).

FIG. 13. The effects of printing parameters on particle geometry. AsCaCl₂ ionic strength increased, the diameter of the printed particlesincreased (A). Increases in the alginate concentration did not changethe particle diameter significantly (B). However, changes in thealginate concentration resulted in dramatic variation in the particlegeometries (C). Most particles printed from 1% alginate showed roundstructures, while most particles generated from 0.5% alginate producedirregular shapes (C). Particles from 2% alginate showed long tailedstructures (C). (*P<0.01, n=10). Magnification: 100×.

FIG. 14. Viability of the cell-loaded micro-particles 1 day afterprinting. The printed particles showed similar mean viability as thecontrols (p>0.05, n=10), which were prepared by manually seedingbeta-TC6 cells onto standard tissue culture plates.

FIG. 15. The insulin secretion profiles for the cultures of beta-TC6cell-containing microparticles and the controls over a 6-day period. Theprinted beta-TC6 cells displayed a continuous insulin secretion duringthis time. The secreted insulin concentrations from the printedparticles were comparable to that of the control group, which wereprepared by manually seeding beta-TC6 cells onto standard tissue cultureplates.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Provided herein and further described below are compositions, devicesand methods useful for the printing of cells and tissues. Thedisclosures of all United States patent references cited herein arehereby incorporated by reference to the extent they are consistent withthe disclosure set forth herein.

As used herein in the description of the invention and the appendedclaims, the singular forms “a,” “an” and “the” are intended to includethe plural forms as well, unless the context clearly indicatesotherwise. Furthermore, the terms “about” and “approximately” as usedherein when referring to a measurable value such as an amount of acompound, dose, time, temperature, and the like, is meant to encompassvariations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specifiedamount. Also, as used herein, “and/or” or “/” refers to and encompassesany and all possible combinations of one or more of the associatedlisted items, as well as the lack of combinations when interpreted inthe alternative (“or”).

A. Printing.

“Printing” as used herein refers to the delivery of individual dropletsof cells and/or compositions with small volumes ranging from 0.5 to 500picoLiters per droplet. In some embodiments, droplets have a volumeranging from 5 to 100 picoLiters per droplet. In other embodiments,droplets range from 10 to 75 picoLiters per droplet. Printing may beperformed by, e.g., using standard printers with print heads that aremodified as described herein. The “print head” is the device in aninkjet printer that sprays droplets (e.g., ink).

Methods and compositions for the inkjet printing of viable cells areknown and described in, for example, U.S. Pat. No. 7,051,654 to Bolandet al.; Wilson et al. (2003) The Anatomical Record Part A 272A: 491-496.The cells may also be printed by other means, such as the methods andcompositions for forming three-dimensional structures by deposition ofviable cells described in U.S. Pat. No. 6,986,739 to Warren et al.

Although not required, cells can typically be printed in the form of a“cell composition” that contains a liquid carrier for the cells. Thecell composition can be in the form of a suspension, solution, or anysuitable form. Examples of suitable liquid carriers include, but are notlimited to, water, ionic buffer solutions (e.g., phosphate buffersolution, citrate buffer solution, etc.), liquid media (e.g., modifiedEagle's medium (“MEM”), Hanks' Balanced Salts, etc.), and so forth. Forinstance, the use of a liquid carrier in the cell composition can ensureadequate hydration and minimize evaporation of the cells after printing.However, the probability of obtaining viable cells in any given printeddrop also decreases with decreasing cell concentration. (T. Boland, USPatent Application Publication No. 20040237822 at para 48)

In some embodiments, cells/compositions are printed with a modifiedinkjet printer. Modifications may include, but are not limited to, meansto control the temperature, humidity, shear force, speed of printing,and firing frequency, by modifications of, e.g., the printer driversoftware and/or the physical makeup of the printer. See, e.g., Pardo etal. (2003) Langmuir 19:1462-1466; U.S. Pat. No. 7,051,654 to Boland etal. Not every modification suggested in these references will besuitable to a given application, as will be appreciated by those skilledin the art. For example, in some embodiments, printers are not modifiedby using new gear mount pillars with closer tolerances by adding ahorizontal support, changing the transistor in the circuit to one withhigher amplification, and reentering the horizontal position encoder.Also, in some embodiments, printer software is not modified to lower theresistive voltages to avoid heating of the solutions above 37° C.

In some embodiments, the inkjet printing device includes atwo-dimensional (X-Y) or three-dimensional (X-Y-Z) plotter (e.g., drivenby the step motors). In some embodiments, the print head is equippedwith a DC solenoid inkjet valve. In some embodiments, a reservoir forloading cell print suspension is connected to the inkjet valve. In someembodiments, the cell print suspension may be supplied from thereservoirs to the inkjet valve by air pressure. In some embodiments, theprint head may be mounted over an X-Y-Z plotter to allow precisedeposition of cells onto a scaffold. Positioning of the XYZ plotterunder the print head may be controlled via a controller. In someembodiments, the controller acquires the positioning information fromsoftware loaded on a computer. In some embodiments, the softwareconverts the image of the target to a four-byte protocol, which is usedto activate specific inkjet valves and coordinate the X-Y-Z position.

In some embodiments, printers (e.g., the commercial printers HP695C andHP550C) may be modified as follows. The printer top cover may be removedand the sensor for the cover disabled. The paper feeding mechanism maybe disabled to allow printing of cells onto solid substrates (e.g.,scaffolds). The ink absorbing pads (which are on the right side of theHP695C and HP550C printers) may be removed (e.g., to avoid the padscontaminating the bottom of the print cartridges during the printingprocess). To offer the capability of the printer to print 3D constructs,a customized z-axis module with a controlled elevator chamber may beadded.

In some embodiments, the inkjet printing device is a thermal bubbleinkjet printer. In general, in a thermal bubble inkjet printer,resistors create heat in the print head, which vaporizes ink to create abubble. As the bubble expands, some of the ink is pushed out of a nozzleonto the paper. A vacuum is created when the bubble collapses, whichpulls more ink into the print head from the cartridge. In the presentinvention, the ink is replaced with, e.g., cells and/or compositions ofinterest (e.g., cells in a liquid carrier), and the paper is replacedwith a suitable substrate, e.g., an agar or collagen coated substrate,or a suitable scaffold. See, e.g., U.S. Pat. No. 6,537,567 to Niklasenet al.

In other embodiments, cells are printed using a piezoelectric crystalvibration print head. In general, a piezoelectric crystal receives anelectric charge that causes it to vibrate, forcing ink out of thenozzle, and pulling more ink into the reservoir. In the presentinvention, the ink is replaced with, e.g., cells and/or a compositionsof interest. Compared with the thermal inkjet printing, the piezo-basedinkjet printing usually requires more power and higher vibrationfrequencies. Typical commercial piezo-printers use frequencies up to 30kHz and power sources ranging from 12 to 100 Watts. Therefore, in someembodiments a piezoelectric crystal vibration print head is used, with avibrating frequency of 1, 5, 10 or 15, to 20, 25, 30, or 35 or more kHz,and power sources from 5, 10, 20, 50, 100, 120, or 150, to 200, 250,300, 350, or 375 or more Watts.

In some embodiments, the print head nozzles are each independentlybetween 0.05 and 200 μm in diameter, or between 0.5 and 100 μm indiameter, or between 10 and 70 μm, or between 20 and 60 μm in diameter.In further embodiments, the nozzles are each independently about 40 or50 μm in diameter. A plurality of nozzles with the same or differentdiameters may be provided. Though in some embodiments the nozzles have acircular opening, other suitable shapes may be used, e.g., oval, square,rectangle, etc., without departing from the spirit of the invention.

As a general guide, eukaryotic animal cells and plant cells aretypically from 10 to 100 μm, and prokaryotic cells are typically from0.1 to 10 μm in diameter. Before printing, in some embodiments the cellsmay be enzymatically dissociated, e.g., from culture plates or explanttissues. Upon enzymatic treatment, the cells typically to shrink tosmaller balls. As a general guide, after enzymatic treatment animalcells are typically from several micrometers to 30 micrometers. Forexample, after trypsin treatment, cells of a porcine aortal endothelialcell line (PAEC cells) are about 10-20 μm.

In some embodiments, the cells/compositions are formulated to provide anencapsulated form upon printing. The encapsulation of cells in permeablecapsules are known and described in, for example, U.S. Pat. No.6,783,964. For example, the cells may be encapsulated in a microcapsuleof from 50 or 100 μm to 1 or 2 mm in diameter that includes an internalcell-containing core of polysaccharide gum surrounded by a semipermeablemembrane; a microcapsule that includes alginate in combination withpolylysine, polyornithine, and combinations thereof. Other suitableencapsulating materials include, but are not limited to, those describedin U.S. Pat. No. 5,702,444.

“Encapsulated” cells are cells or small clusters of cells or tissue thatare surrounded by a selective membrane laminate that allows passage ofoxygen and other required metabolites, releases certain cell secretions(e.g., insulin), but limits the transport of the larger agents of thehost's immune system to prevent immune rejection. Encapsulation may beuseful for, e.g., implanting and/or injecting cells or tissuescontaining living xenogeneic or allogeneic cells while reducing the riskof immune rejection in a host. This may be useful, e.g., to treatdiseases due to inadequate or loss or secretory cell function, orailments that would benefit from the addition of certain secretorycells, e.g., acute liver failure, type I diabetes, chronic pain,Parkinson's disease, etc. Other uses of encapsulated cells as describedherein include, but are not limited to, single cell analysis, highthroughput drug screening, and stem cell differentiation at the singlecell level.

“Microencapsulation” of cells is where one, two, three or several cellsare encapsulated. The microencapsulated cells may be referred to as“microparticles.” In some embodiments, each membrane encapsulates 10cells or less, preferably 5 cells or less, of at least 50, 70, 80, 90 or95% or more of the printed cells.

In some embodiments, three-dimensional arrays are formed.Three-dimensional cell arrays are commonly used in tissue engineeringand biotechnology for in vitro and in vivo cell culture. In general, athree-dimensional array is one which includes two or more layersseparately applied to a substrate, with subsequent layers applied to thetop surface of previous layers. The layers can, in one embodiment, fuseor otherwise combine following application or, alternatively, remainsubstantially separate and divided following application to thesubstrate. Three-dimensional arrays may be formed in a variety of waysin accordance with the present invention. For example, in oneembodiment, three-dimensional arrays may be formed by printing multiplelayers onto the substrate.

The thickness of a printed layer (e.g., cell layer, support layer, etc.)may generally vary depending on the desired application. For example, insome embodiments, the thickness of a layer containing cells is fromabout 2 micrometers to about 3 millimeters, and in some embodiments,from about 20 micrometers to about 100 micrometers. Further, asindicated above, support compounds, such as gels, are often used tofacilitate the survival of printed cells.

When printing certain types of two-dimensional or three-dimensionalarrays, it is sometimes desired that any subsequent cell growth issubstantially limited to a predefined region. Thus, to inhibit cellgrowth outside of this predefined region, compounds may be printed orotherwise applied to the substrate that inhibit cell growth and thusform a boundary for the printed pattern. Some examples of suitablecompounds for this purpose include, but are not limited to, agarose,poly(isopropyl N-polyacrylamide) gels, and so forth. In one embodiment,for instance, this “boundary technique” may be employed to form amulti-layered, three-dimensional tube of cells, such as blood vessels.For example, a cell suspension may be mixed with a first gel (“Gel A”)in one nozzle, while a second gel (“Gel B”) is loaded into anothernozzle. Gel A induces cell attachment and growth, while Gel B inhibitscell growth. To form a tube, Gel A and the cell suspension are printedin a circular pattern with a diameter and width corresponding to thediameter and wall thickness of the tube, e.g., from about 3 to about 10millimeters in diameter and from about 0.5 to about 3 millimeters inwall thickness. The inner and outer patterns are lined by Gel B definingthe borders of the cell growth. For example, a syringe containing Gel Aand “CHO” cells and a syringe containing Gel B may be connected to thenozzle. Gel B is printed first and allowed to cool for about 1 to 5minutes. Gel A and CHO cells are then printed on the agarose substrate.This process may be repeated for each layer.

The present invention includes the printing of tissues by theappropriate combination of cell and support material, or two or three ormore different cell types typically found in a common tissue, preferablyalong with appropriate support compound or compounds, and optionallywith one or more appropriate growth factors. Cells, support compounds,and growth factors may be printed from separate nozzles or through thesame nozzle in a common composition, depending upon the particulartissue (or tissue substitute) being formed. Printing may besimultaneous, sequential, or any combination thereof. Some of theingredients may be printed in the form of a first pattern (e.g., anerodable or degradable support material), and some of the ingredientsmay be printed in the form of a second pattern (e.g., cells in a patterndifferent from the support, or two different cell types in a differentpattern). Again, the particular combination and manner of printing willdepend upon the particular tissue construct desired.

B. Cells.

Any type of cell may be printed using the methods herein, includingprokaryotic and eukaryotic cells. Examples of eukaryotic cells that maybe printed using the methods herein include, but are not limited to,mammalian cells, including stem cells, progenitor cells anddifferentiated cells, without limitation. Stem cells have the ability toreplicate through numerous population doublings (e.g., at least 60-80),in some cases essentially indefinitely, and also have the ability todifferentiate into multiple cell types (e.g., is pluripotent ormultipotent). It is also possible for cells to be transfected with acompound of interest that results in the cells becoming immortalized(i.e., able to double more than 50 times). For example, it has beenreported that mammalian cell transfection with telomerase reversetranscriptase (hTERT) can immortalize neural progenitor cells (See U.S.Pat. No. 7,150,989 to Goldman et al.).

“Embryonic stem cell” as used herein refers to a cell that is derivedfrom the inner cell mass of a blastocyst and that is pluripotent.

“Amniotic fluid stem cell” as used herein refers to a cell, or progenyof a cell, that (a) is found in, or is collected from, mammalianamniotic fluid, mammalian chorionic villus, and/or mammalian placentaltissue, or any other suitable tissue or fluid from a mammalian donor,(b) is pluripotent; (c) has substantial proliferative potential, (d)optionally, but preferably, does not require feeder cell layers to growin vitro, and/or (e) optionally, but preferably, specifically bindsc-kit antibodies (particularly at the time of collection, as the abilityof the cells to bind c-kit antibodies may be lost over time as the cellsare grown in vitro).

“Pluripotent” as used herein refers to a cell that has completedifferentiation versatility, e.g., the capacity to grow into any of theanimal's cell types. A pluripotent cell can be self-renewing, and canremain dormant or quiescent with a tissue. Unlike a totipotent cell(e.g., a fertilized, diploid egg cell) a pluripotent cell cannot usuallyform a new blastocyst.

“Multipotent” as used herein refers to a cell that has the capacity togrow into any of a subset of the corresponding animal cell types. Unlikea pluripotent cell, a multipotent cell does not have the capacity toform all of the cell types of the corresponding animal.

Cells may be syngeneic (i.e., genetically identical or closely related,so as to minimize tissue transplant rejection), allogeneic (i.e., from anon-genetically identical member of the same species) or xenogeneic(i.e., from a member of a different species). Syngeneic cells includethose that are autogeneic (i.e., from the subject to be treated) andisogeneic (i.e., a genetically identical but different subject, e.g.,from an identical twin). Cells may be obtained from, e.g., a donor(either living or cadaveric) or derived from an established cell strainor cell line. For example, cells may be harvested from a donor (e.g., apotential recipient of a bioscaffold graft) using standard biopsytechniques known in the art.

According to some embodiments, at least a portion of the cells areviable after they are printed. “Viable cells” includes cells that adhereto a culture dish or other substrate and/or are capable of survival(e.g., proliferation). In some embodiments, at least 30, 40 or 50% ofthe total cells loaded are viable, and in further embodiments at least60, 70, 80, or 90% or more of the total cells loaded are viable afterprinting. Cell viability may be measured by any conventional means,e.g., the MTS assay, and at a reasonable time after printing, e.g., 1day after printing completion. Viability is measured upon incubationunder conditions known in the art to be optimal for survival of thecertain cells types present. For example, many eukaryotic cell types aretypically incubated in a suitable medium at 5% carbon dioxide (95%atmospheric air) and 37 degrees Celsius.

Various mechanisms may be employed to facilitate the survival of thecells during and/or after printing. Specifically, compounds may beutilized that support the printed cells by providing hydration,nutrients, and/or structural support. These compounds may be applied tothe substrate using conventional techniques, such as manually, in a washor bath, through vapor deposition (e.g., physical or chemical vapordeposition), etc. These compounds may also be combined with the cellsand/or compositions before and/or during printing, or may be printed orotherwise applied to the substrate (e.g., coated) as a separate layerbeneath, above, and/or between cell layers. For example, one suchsupport compound is a gel having a viscosity that is low enough underthe printing conditions to pass through the nozzle of the print head,and that can gel to a stable shape during and/or after printing. Suchviscosities are typically within the range of from about 0.5 to about 50centipoise, in some embodiments from about 1 to about 20 centipoise, andin some embodiments, from about 1 to about 10 centipoise. Some examplesof suitable gels that may be used in the present invention include, butare not limited to, agars, collagen, hydrogels, etc.

Another polymer used for hydrogels is alginate, a natural polysaccharideextracted from seaweed. One feature of alginate solutions is theirgelling properties in the presence of divalent cations (e.g., Mg++,Ca++, Sr++, Ba++).

Besides gels, other support compounds may also be utilized in thepresent invention. Extracellular matrix analogs, for example, may becombined with support gels to optimize or functionalize the gel. In someembodiments, one or more growth factors may also be introduced in theprinted arrays. For example, slow release microspheres that contain oneor more growth factors in various concentrations and sequences may becombined with the cells and/or composition. Other suitable supportcompounds might include those that aid in avoiding apoptosis andnecrosis of the developing structures. For example, survival factors(e.g., basic fibroblast growth factor) may be added. In addition,transient genetic modifications of cells having antiapoptotic (e.g.,bcl-2 and telomerase) and/or blocking pathways may be included incompositions printed. Adhesives may also be utilized to assist in thesurvival of the cells after printing. For instance, soft tissueadhesives, such a cyanoacrylate esters, fibrin sealant, and/orgelatin-resorcinol-formaldehyde glues, may be utilized to inhibitnascent constructs from being washed off or moved following the printingof a layer. In addition, adhesives, such as arginine-glycine-asparticacid (RGD) ligands, may enhance the adhesion of cells to a gellingpolymer or other support compound. Extracellular proteins, extracellularprotein analogs, etc., may also be utilized.

“Growth factor” may be any naturally occurring or synthetic growthfactor, including combinations thereof, suitable for the particulartissue or array being printed. Numerous growth factors are known.Examples include, but are not limited to, insulin-like growth factor(e.g., IGF-1), transforming growth factor-beta (TGF-beta),bone-morphogenetic protein, fibroblast growth factor, platelet derivedgrowth factor (PDGF), vascular endothelial growth factor (VEGF),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), epidermal growth factor, fibroblast growth factor (FGF) (numbers1, 2 and 3), osteopontin, bone morphogenetic protein-2, growth hormonessuch as somatotropin, cellular attractants and attachment agents, etc.,and mixtures thereof. See, e.g., U.S. Pat. Nos. 7,019,192; 6,995,013;and 6,923,833. For example, growth factor proteins may be provided inthe printed composition and/or encoded by plasmids transfected intoprinted cells.

In some embodiments, cells, compositions, support compounds, and/orgrowth factors may be printed from separate nozzles or through the samenozzle in a common composition, depending upon the particular tissue (ortissue substitute) being formed. Printing may be simultaneous,sequential, or any combination thereof. Some of the ingredients may beprinted in the form of a first pattern (e.g., an erodable or degradablesupport material), and some of the ingredients may be printed in theform of a second pattern (e.g., cells in a pattern different from thesupport, or two different cell types in a different pattern). Theparticular combination and manner of printing will depend upon theparticular tissue being printed.

In some embodiments, cells/compositions are printed onto a substrate,e.g., a biocompatible scaffold, which may be subsequently implanted intoa subject in need thereof. In other embodiments, cells/compositions ofinterest are directly printed in vivo onto living tissues in the body,with or without prior substrate application (e.g., a layer of fibrin) inwhich the cells may attach.

“Isolated” as used herein signifies that the cells are placed intoconditions other than their natural environment. Tissue or cells are“harvested” when initially isolated from a subject, e.g., a primaryexplant.

The “primary culture” is the first culture to become established afterseeding disaggregated cells or primary explants into a culture vessel.“Expanding” or “expansion” as used herein refers to an increase innumber of viable cells. Expanding may be accomplished by, e.g.,“growing” the cells through one or more cell cycles, wherein at least aportion of the cells divide to produce additional cells. “Growing” asused herein includes the culture of cells such that the cells remainviable, and may or may not include expansion and/or differentiation ofthe cells.

“Passaged in vitro” or “passaged” refers to the transfer or subcultureof a cell culture to a second culture vessel, usually implyingmechanical or enzymatic disaggregation, reseeding, and often divisioninto two or more daughter cultures, depending upon the rate ofproliferation. If the population is selected for a particular genotypeor phenotype, the culture becomes a “cell strain” upon subculture, i.e.,the culture is homogeneous and possesses desirable characteristics(e.g., the ability to express a certain protein or marker).

“Express” or “expression” of a protein or other biological marker meansthat a gene encoding the same of a precursor thereof is transcribed, andpreferably, translated. Typically, according to the present invention,expression of a coding region of a gene will result in production of theencoded polypeptide, such that the cell is “positive” for that proteinor other downstream biological marker.

“Cartilage cells” include those cells normally found in cartilage, whichcells include chondrocytes. “Chondrocytes” produce and maintain theextracellular matrix of cartilage, by, e.g., producing collagen andproteoglycans. Cartilage is a highly specialized connective tissue foundthroughout the body, and its primary function is to provide structuralsupport for surrounding tissues (e.g., in the ear and nose) or tocushion (e.g., in the trachea and articular joints). Types of cartilageinclude hyaline cartilage (articular joints, nose, trachea,intervertebral disks (NP), vertebral end plates), elastic cartilage(tendon insertion site, ligament insertion site, meniscus,intervertebral disks (AP)), costochondral cartilage (rib, growth plate),and fibrocartilage (ear). The loss of cartilage in a subject can beproblematic, as it has a very limited repair capacity. “Mesenchymal stemcells” or “MSCs” are progenitors of chondrocytes. MSCs can alsodifferentiate into osteoblasts. Cartilage cells/tissues produced by theprocesses described herein are useful for, among other things,implantation into a subject to treat cartilage injury or disease.

“Bone cells” include those cells normally found in bone, and includeosteoblasts, osteoclasts, osteocytes, and any combination thereof. Bonecells/tissues produced by the processes described herein are useful for,among other things, implantation into a subject to treat bone fracturesor defects, and/or promote bone healing.

“Muscle cells” include those cells normally found in muscle tissue,including smooth muscle cells, cardiac muscle cells, skeletal musclecells, and any combination thereof. Muscle cells/tissues produced by theprocesses described herein are useful for, among other things,implantation into a subject to treat muscle injuries or defects, and/orpromote muscle healing.

“Skin cells” include those cells normally found in skin, and includeepidermal cells (e.g., keratinocytes, melanocytes, Merkel cells,Langerhan cells, etc., and any combination thereof) and dermal cells(e.g., fibroblasts, adipocytes, mast cells, macrophages, and anycombination thereof). Skin tissue produced by the process of the presentinvention is useful for implantation into or on a subject to, forexample, treat burns, and other wounds such as incisions, lacerations,and crush injuries (e.g., postsurgical wounds, and posttraumatic wounds,venous leg ulcers, diabetic foot ulcers, etc.)

“Pancreatic cells” include those cells normally found in the pancreas,and include pancreatic islet cells, e.g., glucagon-synthesizing A (α)cells, insulin-producing B (β) cells, D (δ) cells, etc., and anycombination thereof. Pancreatic islet tissue produced by the processesdescribed herein is useful for, among other things, implantation into asubject to treat diabetes (including type I and type II diabetes).

“Kidney cells” include those cells normally found in the kidney, andinclude interstitial cells (e.g., interstitial peritubular cells whichsecrete erythropoietin), endothelial cells, etc., or any combinationthereof.

“Nervous system cells” or “nerve cells” include those cells normallyfound in the central and/or peripheral nervous system, includingneuronal cells (e.g., cortical neurons, hippocampal neurons,dopaminergic neurons, cholinergic neurons, adrenergic neurons,noradrenergic neurons, etc., including any combination thereof), andglial cells (e.g., neuroglia, astrocytes, oligodendrocytes, Schwanncells, etc., including any combination thereof). Nerve cells produced bythe processes described herein is useful, among other things, forimplantation into a subject to treat nerve injury or degenerativediseases such as Parkinson's disease and Alzheimer's disease.

“Liver cells” include those cells normally found in the liver, andinclude hepatoblasts, hepatocytes, hepatic stellate cells, Kupffercells, sinusoidal endothelial cells, etc., including any combinationthereof. Livers cells produced by the processes described herein isuseful, among other things, for implantation into a subject to treatacute or chronic liver disease.

In some embodiments stem cells are printed onto substrates by inkjetprinting. Stem cells may be printed alone (typically in combination witha support compound or compounds) or in combination with one or moreadditional cells (e.g., in a combination selected to produce a tissue asdescribed above). In some embodiments, stem cells are differentiatedinto cells of interest.

“Differentiation” and “differentiating” as used herein include (a)treatment of the cells to induce differentiation and completion ofdifferentiation of the cells in response to such treatment, both priorto printing on a substrate, (b) treatment of the cells to inducedifferentiation, then printing of the cells on a substrate, and thendifferentiation of the cells in response to such treatment after theyhave been printed, (c) printing of the cells, simultaneously orsequentially, with a differentiation factor(s) that inducesdifferentiation after the cells have been printed, (d) contacting thecells after printing to differentiation factors or media, etc., andcombinations of all of the foregoing. In some embodimentsdifferentiation may be modulated or delayed by contacting an appropriatefactor or factors to the cell in like manner as described above. In someembodiments appropriate differentiation factors are one or more of thegrowth factors described above. Differentiation and modulation ofdifferentiation can be carried out in accordance with known techniques,e.g., as described in U.S. Pat. No. 6,589,728, or U.S. PatentApplication Publication Nos.: 2006006018 (endogenous repair factorproduction promoters); 20060013804 (modulation of stem celldifferentiation by modulation of caspase-3 activity); 20050266553(methods of regulating differentiation in stem cells); 20050227353(methods of inducing differentiation of stem cells); 20050202428(pluripotent stem cells); 20050153941 (cell differentiation inhibitingagent, cell culture method using the same, culture medium, and culturedcell line); 20050131212 (neural regeneration peptides and methods fortheir use in treatment of brain damage); 20040241856 (methods andcompositions for modulating stem cells); 20040214319 (methods ofregulating differentiation in stem cells); 20040161412 (cell-based VEGFdelivery); 20040115810 (stem cell differentiation-inducing promoter);20040053869 (stem cell differentiation); or variations of the above orbelow that will be apparent to those skilled in the art.

Generally, when cells of the invention are used for treating a subject,e.g., encapsulated cells, the cells are formulated into a pharmaceuticalcomposition containing the cells in admixture with a pharmaceuticallyacceptable vehicle or carrier. Such formulations can be prepared usingtechniques well known in the art. See, e.g., U.S. Patent Application2003/0180289; Remington: The Science and Practice of Pharmacy, AlfonsoR. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins:Philadelphia, Pa., 2000. In the manufacture of a pharmaceuticalformulation according to the invention, the cells are typically admixedwith, inter alia, an acceptable carrier. The carrier must, of course, beacceptable in the sense of being compatible with any other ingredientsin the formulation and must not be deleterious to the patient. Thecarrier may be a solid or a liquid, or both (e.g., hydrogels), and canbe formulated with the cells as a unit-dose formulation. In oneembodiment the cells are provided as a suspension in the carrier toreduce clumping of the cells.

“Implant” refers to a product configured to repair, augment or replace(at least a portion of) a natural tissue of a subject, e.g., forveterinary or medical (human) applications. The term “implantable” meansthe device can be inserted, embedded, grafted or otherwise chronicallyattached or placed on or in a patient. Implants include, but are notlimited to, a “scaffold” or “bioscaffold” (which may or may not furthercomprise cells seeded onto the scaffold or bioscaffold).

“Subjects” are generally human subjects and include, but are not limitedto, “patients.” The subjects may be male or female and may be of anyrace or ethnicity, including, but not limited to, Caucasian,African-American, African, Asian, Hispanic, Indian, etc. The subjectsmay be of any age, including newborn, neonate, infant, child,adolescent, adult and geriatric subjects.

Subjects may also include animal subjects, particularly vertebratesubjects, e.g., mammalian subject such as canines, felines, bovines,caprines, equines, ovines, porcines, rodents (e.g., rats and mice),lagomorphs, non-human primates, etc., or fish or avian subjects, for,e.g., veterinary medicine and/or research or laboratory purposes.

“Treat” refers to any type of treatment that imparts a benefit to asubject, e.g., a patient afflicted with a trauma or a disease. Forexample, arthritis is a disease that affects cartilage. Treatingincludes actions taken and actions refrained from being taken for thepurpose of improving the condition of the patient (e.g., the relief ofone or more symptoms), delay in the onset or progression of the disease,etc. In some embodiments, treating includes reconstructing cartilagetissue (e.g., where such tissue has been damaged or lost by injury ordisease) by implanting a scaffold into a subject in need thereof.Scaffolds may be implanted, e.g., at or adjacent to the site of injury,and/or at another site in the body of a subject that would impart abenefit to the subject, as would be appreciated by one of skill in theart.

C. Scaffolds.

“Scaffold” or “bioscaffold” refers to an array of natural and/orsynthetic matrix molecules to which cells or fibers can attach. Thefibers may include extracellular matrix molecules or components, such aselastin, elastic strands or peptides, fibrin, collagen, proteoglycans,hyaluronan or hyaluronan oligomers, synthetic fibers or fibrils, orbioactive hydrogels, microparticles, beads, liposomes, or vesicles.Scaffolds may further include extracellular matrix components, such aselastin, elastin-like or elastin-mimetic peptides, fibrin,proteoglycans, commercially available matrix or matrix-substitutes suchas Matrigel™ matrix (BD Biosciences, San Jose, Calif., USA), collagen ofany type, synthetic fibers or fibrils, and/or hydrogels.

Collagens are found throughout the body, and are of at least 12 types(type I-XII). As an example, the primary type of collagen found inarticular cartilage is type II, followed by type IX and type XI.

A “biodegradable scaffold,” “biodegradable mesh” or “biodegradablematrix” is a scaffold having materials capable of being degraded and/orabsorbed by a subject's body. Desirably, the scaffold or matrix isporous to allow for cell deposition both on and in the pores of thematrix, and, in certain embodiments, is shaped. Such formulations can beprepared by supplying at least one cell population to a biodegradablescaffold to seed the cell population on and/or into the scaffold. Insome embodiments, the seeded scaffold is then implanted in the body ofthe recipient subject, where the organized cell populations facilitatethe formation of functional tissue structures.

Biodegradable materials that may be used include, e.g., naturalpolymers, such as collagen and elastin, and/or synthetic polymers, whichcan be degraded, e.g., by hydrolysis, at a controlled rate and arereabsorbed. Examples of other suitable materials are provided in U.S.Pat. No. 7,186,554, which is incorporated by reference herein.

Examples of biodegradable synthetic polymers include, but are notlimited to, poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,poly(lactic acid-co-glycolic acid)s, poly(caprolactone)s,polycarbonates, polyesteramides, polyanhydrides, poly(amino acid)s,poly(ortho ester)s, polycyanoacrylates, polyamides, polyacetals,poly(ether ester)s, copolymers of poly(ethylene glycol)s and poly(orthoester)s, poly(dioxanone)s, poly(alkylene alkylate)s, biodegradablepolyurethanes, as well as copolymers thereof. See, e.g., U.S. Pat. No.7,097,857, which is incorporated by reference herein. Any polymer orcopolymer may be combined or blended by methods known in the art.

For example, poly(lactide)s include, but are not limited to,poly(lactide) (also known as polylactic acid, or PLA) such aspoly(_(L)-lactide) (PLLA), poly(_(D,L)-lactide) (PDLLA), and copolymersthereof. Copolymers of poly(lactide) include, but are not limited to,poly(lactide-co-glycolide) (PLGA), which is a copolymer of PLA withpolyglycolide (PGA) (e.g., poly(_(D,L)-lactide-co-glycolide)); andpoly(lactide-co-caprolactone) (PLCL), which is a copolymer of PLA withpoly(caprolactone) (PCL).

Examples of poly(caprolactone)s include, but are not limited to,poly(ε-caprolactone). Copolymers of PCL include, but are not limited to,poly(lactide-co-caprolactone) (PLCL), etc.

In some embodiments, cells and/or substrates may be oriented withrespect to one or more axes. “Oriented” cells and/or substratestypically have one (or more) axis of orientation (e.g., longitudinalaxis), which may be in any desired direction within the region ofinterest. It will be appreciated that “orienting” as used herein mayinclude partial or total orientation, so long as a sufficient increasein organization is achieved to produce the effect or benefit intendedfor the particular implementation of the method described herein. Forexample, fibers and/or cells may be oriented along a longitudinal axissuch that greater than 70, 80, 90, or 95% or more of the fibers and/orcells are at an angle of 50, 40, 30, 20, or 10 degrees or less from thereference axis in any direction.

“Anisotropic” means that the physical properties (e.g., elasticity,tensile strength, elongation at break, etc.) of a material (e.g.,myotube, scaffold, etc.) are different depending upon the direction ofaction (e.g., stretch or strain), as opposed to “isotropic,” in whichthe properties of a material are identical in all directions. Forexample, an anisotropic cell substrate may have a greater ultimatetensile strength along one axis (e.g., the longitudinal axis) than alongan axis perpendicular to the axis (e.g., by 0, 1 or 2 to 4, 5, 6 or moreMPa measured under wet condition at room temperature). The elongation atbreak may be smaller along one axis (e.g., the longitudinal axis) thanalong an axis perpendicular to the axis (e.g., by 10, 20, 30 or 40 to50, 60, 70 or 80% or more). The peak of a stress curve (MPa) may bereached at a lower strain (%) along one axis (e.g., the longitudinalaxis) as compared to an axis perpendicular to the axis.

In some embodiments, the biodegradable scaffold can be “shaped” usingmethods such as, for example, solvent casting, compression molding,filament drawing, meshing, leaching, weaving and coating. In solventcasting, a solution of one or more polymers in an appropriate solvent,such as methylene chloride, is cast as a branching pattern reliefstructure. After solvent evaporation, a thin film is obtained. Incompression molding, a polymer is pressed at pressures up to 30,000pounds per square inch into an appropriate pattern. Filament drawinginvolves drawing from the molten polymer and meshing involves forming amesh by compressing fibers into a felt-like material. In leaching, asolution containing two materials is spread into a shape that resemblesthe final form. Next, a solvent is used to dissolve away one of thecomponents, resulting in pore formation (see U.S. Pat. No. 5,514,378).In nucleation, thin films in the shape of a reconstructive graft areexposed to radioactive fission products that create tracks of radiationdamaged material. Next the polycarbonate sheets are etched with acid orbase, turning the tracks of radiation-damaged material into pores.Finally, a laser may be used to shape and burn individual holes throughmany materials to form a reconstructive graft structure with uniformpore sizes. These shaping techniques may be employed in combination. Forexample, a biodegradable matrix can be weaved, compression molded andalso glued. Furthermore, different polymeric materials shaped bydifferent processes may be joined together to form a composite shape.The composite shape can be a laminar structure. For example, a polymericmatrix can be attached to one or more polymeric matrixes to form amultilayer polymeric matrix structure. The attachment can be performedby gluing with a liquid polymer or by suturing. In addition, thepolymeric matrix can be formed as a solid block and shaped by laser orother standard machining techniques to its desired final form. Lasershaping refers to the process of removing materials using a laser.

The biodegradable scaffold can be treated with additives or drugs priorto implantation (before or after it is seeded with cells), e.g., topromote the formation of new tissue after implantation. Thus, forexample, growth factors, cytokines, extracellular matrix components,and/or other bioactive materials can be added to the biodegradablescaffold to promote graft healing and the formation of new tissue. Suchadditives will in generally be selected according to the tissue or organbeing reconstructed or augmented, to ensure that appropriate new tissueis formed in the engrafted organ or tissue. For examples of suchadditives for use in promoting bone healing, see, e.g., Kirker-Head(1995) Vet. Surg. 24 (5):408-19.

D. Electrospinning.

In some embodiments, scaffolds are formed by electrospinning.“Electrospinning” is a fiber spinning technique driven by a high voltageelectrostatic field using a polymer solution that produces fibers withdiameters ranging from several micrometers down to 100 nm or less. Insome embodiments, fibers have an average diameter from 10, 100, or 200nm to 400, 500, 750 or 1000 nm. The nano-scaled structure of theelectrospun scaffolds can support cell adhesion and guide theirbehavior. Moreover, the composition, structure, and mechanicalproperties of biomaterials can be controlled. See, e.g., Li et al.,“Electrospinning of Nanofibers: Reinventing the Wheel?” AdvancedMaterials (2004) vol. 16, no. 4, pp. 1151-1170. See also U.S. Pat. Nos.7,326,043; 7,323,425; 7,172,765, each of which is incorporated byreference herein.

To avoid the potential adverse effect caused by static discharge of highvoltage on other devices (e.g., a controller), in some embodiments anapparatus useful for electrospinning includes means for conductiveisolation of the high voltage.

In some embodiments, a device is provided that is configured for bothelectrospinning and inkjet printing. In some embodiments, the inkjetprinting platform includes an XYZ plotter driven by the step motors andthe print head equipped with a DC solenoid inkjet valve. The print headmay be mounted over a XYZ plotter platform to allow precise depositionof cells onto a scaffold generated by electrospinning. In someembodiments, positioning the XYZ plotter under the print head iscontrolled via a controller, which acquires the positioning informationfrom software loaded on a computer. This software converts the image ofthe target to a special four byte protocol, which is used to activatespecific inkjet valve and coordinate X-Y-Z position. In someembodiments, scaffolds are spun and/or cells are printed directly into asuitable liquid (e.g., media).

According to some embodiments, the electrospinning apparatus is used togenerate polymeric fiber-based scaffolds. In some embodiments, anelectrospinning head is also mounted over the XYZ plotter (e.g., intandem with the inkjet head). In some embodiments, the high voltagepower supply, used to provide a high voltage field for electrospinning,is modulated by the same controller as the XYZ plotter.

In order to avoid the potential adverse effects caused by staticdischarge of high voltage on the logic circuitry of the customizedcontroller, according to some embodiments the controller is conductivelyisolated from the high voltage power supply.

In some embodiments, “conductive isolation” may include the use ofwireless communication. Wireless communication includes, but is notlimited to, radio frequency communications including, for example, anear field communication (NFC) protocol, Bluetooth, and/or Wifi, amongothers. Some embodiments may include one or more transceivers coupled tothe electro-spinning print head and/or the controller.

In some embodiments, conductive isolation may include may include fiberoptics communication. Fiber optics may use light transmission to receiveand/or send the data between the control system and the high voltagesupply. Some embodiments may include optical encoders and/or decoders.

In some embodiments, the scaffold is created “in sequence”layer-by-layer, with an electrospun layer (A), then a printed layer (B)in series, such as:

ABABABABABABABA

Each layer may include one or more individual layers of the samecomposition, e.g., layer A includes more than one layer of electrospunmaterial, layer B includes more than one layer of an inkjet printedcomposition, etc. For example, a layer “B” may include individual layersof different cell types that together form a tissue.

The use of a layer-by-layer arrangement according to some embodimentscan provide adequate mechanical properties for implanting a scaffoldconstruct into a subject. For example, according to some embodiments,the scaffold is capable of being sutured as part of implantationsurgery. Non-woven electrospun fibers can be spun to have controlledarchitectures, including aligned fibers, fibers having certain angleswith respect to one or more axes, etc., as needed to more preciselymimic the natural environment of the tissue being created.

The present invention is explained in greater detail in the followingnon-limiting examples.

Example 1 Printing Cartilage Constructs Using a Hybrid Printing Device

To demonstrate the feasibility of generating structured cartilageconstructs using a combination of electrospinning and inkjet printing, alayer-by-layer electrospun PCL and inkjet printed chrondrocyte scaffoldwas created (FIG. 1 and FIG. 2).

Electrospinning was first used to fabricate the PCL scaffold layer withpolymeric nanofibers. Then the inkjet print head laid down rabbitelastic chondrocytes with the fibrin hydrogels on the electro-spunlayer. By alternately applying electrospinning and inkjet printing, a 3Dcartilage construct containing multi-layers of cells and scaffolds wasgenerated (FIG. 3). The multi-layered structures of the constructs wereobserved under SEM examination (FIG. 4). Printed chondrocytes attachedto the collagen/elastin within the layer (FIG. 5).

Mechanical properties, cell viability, and cartilage production of theprinted constructs were evaluated. The fabricated scaffolds demonstratedmore stiffness and were able to withstand greater tensile stress thanalginate hydrogels (FIG. 6). Over 81% of the cells within the constructmaintained viability after printing. Histological analysis showedcartilage-specific ECM production (e.g. GAGs and type II and IVcollagens) both in vitro (FIG. 7) and in vivo (FIG. 9), indicating theformation of cartilage tissues. In vivo magnetic resonance imaging (MRI)showed cartilage taking shape after 2-week implantation into nude mice(FIG. 8).

Materials and Methods.

Polycaprolactone (PCL) (Mn 42,500) and Pluronic F-127 were obtained fromSigma-Aldrich (St. Louis, Colo.) and acetone from Fisher Chemicals (FairLawn, N.J.). Bovine fibrinogen and bovine thrombin were obtained fromSigma-Aldrich and rat tail. PCL (10% wt/wt) and Pluronic F-127 (5% w/w)were dissolved in acetone under gentle stir in a warm water bath (50°C.). The Pluronic F-127 in the polymer solution was used.

Chondrocytes were obtained from rabbits' ear cartilage as previouslydescribed (E. Sanz, L. Penas, J. L. Lequerica, Plast Reconstr Surg 2007,119, 1707). Briefly, after sedation of the rabbit, the ear was shaved,cleaned with povidone-iodine, and draped. A pocket was resected understerile conditions at the subperichondrial level and an approximately2×2-cm piece of cartilage was removed. Biopsy specimens were washed inphosphate-buffered saline (Gibco-BRL, Grand Island, N.Y.) and finelyminced under sterile conditions. Chondrocytes were released from thecartilage by enzymatic digestion with collagenase type B (BoehringerMannheim GmbH, Germany). The minced cartilage was placed in tissueculture dishes containing 10 to 12 ml of Ham's F12 medium withglutamine/bicarbonate supplemented with 10% fetal bovine serum, 100 U/mlpenicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin B(Gibco-BRL), and 1 mg/ml collagenase type B and incubated at 37° C. inan orbital shaker (Stuart Scientific, Surrey, United Kingdom). After 24hours, the undigested pieces were discarded and the supernatant wasseeded onto tissue culture dishes. Each dish was filled with 20 ml offresh culture media and incubated in a humidified incubator at 37° C.with 5% CO2. Media was changed every 3 or 4 days. Culture medium wasDulbecco's modified eagle medium (DMEM) supplemented with 10% fetalbovine serum (FBS), 1% antibiotics, and 1% glutamine.

Rabbit chondrocytes were collected from the culture. After trypsinizing,cell pellets were collected and re-suspended in the mixture offibrinogen (10 mg/ml)/collagen (1.5 mg/ml) in 1× phosphate buffer saline(PBS, Sigma-Aldrich) with the final concentration of 3-4×10⁶ cells/ml.

Hybrid Printing System.

To build cartilage constructs, we have developed a hybrid printingsystem by incorporating the electrospinning apparatus into the inkjetprinting platform. FIG. 10 shows the schematic drawing of this system.

The inkjet printing platform is composed of a customized XYZ plotterdriven by the step motors and the print head equipped with a DC solenoidinkjet valve (Offshore Solutions Inc, Beaverton, Oreg.). A reservoir forloading cell print suspension is connected to the inkjet valve. By airpressure, the cell print suspension is supplied to the inkjet valve fromreservoirs. The print head is mounted over a XYZ plotter platform toallow precise deposition of cells onto a scaffold generated byelectrospinning. Positioning the XYZ plotter under the print head iscontrolled via a customized controller. The controller acquires thepositioning information from software loaded on a computer. Thissoftware converts the image of the target to a special four byteprotocol, which is used to activate specific inkjet valve and coordinateX-Y-Z position.

In this hybrid system, the electrospinning apparatus is used to generatepolymeric fibers based scaffolds. An electrospinning head, in tandemwith the inkjet head, is also mounted over the XYZ plotter. The highvoltage power supply, used to provide a high voltage field forelectrospinning, is modulated by the same customized controller.

To avoid the potential adverse effect caused by static discharge of highvoltage on the logic circuitry of the customized controller, it isrequired to conductively isolate the controller from the high voltagepower supply. For this purpose, we used fiber optics or Bluetooth toconnect the control system to the high voltage power supply. The fiberoptics use light transmission to receive or send the data between thecontrol system and the high voltage power supply. In this way, the twosystems can be effectively isolated.

Fabrication of Cartilage Constructs.

A layer of PCL was spun for 30 min at 3 mL/hr, and then the 3 mL of theprint suspension containing cells and the fibrinogen/collagen mixturewas printed through the inkjet valve onto the electro-spun PCL scaffoldlayer to form a cell/matrix layer. 1.5 mL thrombin (20 UI/ml dissolvedin PBS) was subsequently printed on the cell/matrix layer to create atransient clot for structural integrity while spinning. Subsequently,PCL was spun for another 10 min at 3 mL/hr, and then inkjet printing ofcell suspension was repeated. A final layer of PCL was spun for 30 minat 3 mL/hr.

Cell Viability.

The viability of chondrocytes within the fabricated cartilage constructs1 days after culture was evaluated by a two-color fluorescence live/deadassay using a solution consisting of 2 μM calcein AM and 4 μM ethidiumhomodimer (EthD-1; Molecular Probes, OR) in 10 ml Phosphate BufferedSaline (PBS). Printed samples with chondrocytes were rinsed to removeresidual serum, and 2 ml of viability testing solution was added to eachsample. The samples were incubated for 30 min at 37° C. followed byrinse with PBS. The samples were viewed using a fluorescent microscopeand the viability of the cells was evaluated by counting the number ofcells stained with calcein AM (green), and this number was compared tothe total number of cells. 81.58+/−3.46% of the cells were alive afterone week in vitro.

Mechanical Properties.

The constructs without cells fabricated by the hybrid printing methodwere mechanically loaded using a uniaxial load frame (InstronCorporation, Issaquah, Wash.). A short segment from the scaffold wasclamped at its cut ends for the axial test. The crosshead speed was setat 0.2 mm/s, and the test was stopped when the force decreased by 10%after the onset of failure. Young's modulus (modulus of elasticity) wascalculated from the slope of the initial linear segment of thestress-strain curve at maximum stress. Ultimate tensile stress (UTS,MPa) at break was calculated as the maximal load recorded during eachtest.

As shown in Table 1, mechanical testing demonstrated that the fabricatedhybrid scaffold has more stiffness and is able to withstand greaterstress than alginate and PCL.

TABLE 1 Mechanical Testing of Fabricated Scaffolds Alginate PCT ScaffoldYoung's Modulus 0.409 ± 0.060 0.709 ± 0.215 1.764 ± 0.704 (MPa) UltimateTensile 0.241 ± 0.082 0.913 ± 0.165 1.113 ± 0.124 Strength (MPa)Elasticity 0.421 ± 0.074 1.095 ± 0.089 0.552 ± 0.209

Microscopy.

The microstructures of the cartilage constructs was evaluated by usingScanning electron microscopy. Cross-sectional electron-micrographs wereobtained at 25.0 kV, 50 Pa, 500× magnification using a Hitachi S-2600Scanning Electron Microscope (Hitachi High Technologies America,Pleasanton, Calif.). Microscopy revealed that the layered structure wasmaintained (FIG. 4). The microstructure of the PCL was also examined anhigh magnification (4000×), and was seen to be fibrous with beads. SEManalysis also demonstrated attachment of chondrocytes to the matrix andextracellular matrix deposition (FIG. 5).

In Vivo Evaluation.

All animal procedures were performed according to the protocols set bythe Wake Forest University Health Sciences Animal Care and UseCommittee. The fabricated cartilage constructs were implantedsubcutaneously into the back of outbred athymic nude (nu/nu) mice(Charles River Laboratories). Four printed constructs were implanted permouse. The samples were retrieved after 8 weeks of implantation andevaluated.

MRI characterization of implanted tissues on animals was performed at 2and 10 weeks after implantation. Experiments were performed using a 7Tsmall animal MRI scanner (Bruker Biospin Inc., Billerica, Mass.), withan actively-shielded gradient set capable of a maximum gradient of 400mT/m. A custom-made Litz volume coil with 25 mm ID (Doty ScientificInc., Columbia, S.C.) was used for both signal transmission andreception. The animals were anesthetized with 3% isoflurane and oxygenat a flow rate of 3 L/min initially, and then maintained with a mixtureof 1.5% isoflurane and oxygen at a flow rate of 1 L/min through a nosecone while in the scanner. The respiration and ECG of the animals weremonitored (SA Instruments Inc, Stoney Brook, N.Y.) throughout the scan.Cartilage implants were identified on T2-weighted multi-slice fastspin-echo Rapid Acquisition with Relaxation Enhancement (RARE) imageswith the following parameters: TR/TE=2500/42 ms, number of echoes, 4, 2NEX, image matrix 256×256, slice thickness 0.6 mm, field of view (FOV) 4cm.

Histology.

The printed samples were both cultured for four weeks in vitro andimplanted for 8 weeks in vivo were histologically evaluated. The printedimplants were surgically removed from the implantation sites 8 weeksafter implantation. All samples were fixed overnight with 10% formalin.After being embedded in paraffin, these samples were cut into 3-5 μmthick sections. Cartilage production was determined by a Masson'sTrichrome (MT) stain for collagen and a Safranin O (Saf O) stain forglycosaminoglycans. In order to identify different collagen typesproduced within the constructs, the printed samples were immunostainedwith anti-Type II collagen antibodies and anti-Type IV collagenantibodies (Dako, Denmark).

Histological analysis of the in vitro cultured samples showed that thechondrocytes maintained their phenotype and were able to producecartilage after 4 weeks. Analysis of sections taken after 2 weeks invitro showed the initiation of cartilage tissue formation (not shown).The cartilage construct at 4 weeks demonstrated a significant populationof chondrocytes as well as deposition of cartilage tissue matrix (FIG.7). Masson's Trichrome staining showed bands of collagen accumulatedaround the cells. Safranin O staining demonstrated the production ofglycosaminoglycans. In cartilage, the principle GAGs are chondroitinsulfate and keratan sulfate, which are markers of ECM deposition.Immunohistochemical analysis of the deposited collagen revealed bothtype II and IV collage, which are major components of cartilage (FIG.7). Control samples demonstrated little, if any, collagen and GAGs.

The printed samples developed into cartilage-like constructs in vivo.Histological analysis showed that printed chondrocytes maintained theirphenotypic characteristics and formed cartilage tissue 8 weekspost-implantation in vivo. Masson's Trichrome staining showed collagenproduction by the cells in vivo (FIG. 9). Similarly, safranin O stainingconfirmed the production of glycosaminoglycans. Control samples failedto produce any collagen or GAGs (data not shown).

Compared to the in vitro conditions, larger amounts of collagen and GAGwere seen in the implant. Moreover, in the implant, the typicalcartilage lacunae structure was found in the samples, and manychondrocytes situated in these lacuna. Taken together, these datasuggest that more mature cartilage tissues were developed in the in vivothan in the in vitro conditions.

Example 2 High-Throughput Production of Single Cell Microparticles UsingInkjet Printing

An insulin-producing beta cell line (TC6) was obtained from AmericanType Culture Collection (ATCC, Manassas, Va.). The cells were maintainedin Dulbecco's Modified Eagles Medium (ATCC) supplemented withheat-inactivated 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.),100 IU of penicillin (Invitrogen) and 100 μg/ml of streptomycin(Invitrogen) and incubated at 37° C. in a humidified 5% CO₂ atmosphere.

Sodium alginate (MVG; Pronova Biomedical, Oslo, Norway) with an overallgluronic acid (G-block) content of 70% (as reported by the manufacturer)was mixed with PBS at different concentrations (0.5%, 1%, and 2%solution (w/v)) and autoclaved. Beta-TC6 cells were trypsinized, cellpellets collected and re-suspended in sodium alginate solutions atdifferent final cell concentrations (2×10⁶, 6×10⁶, and 12×10⁶ cells/ml).

HP DeskJet 550C printers were modified using previously describedmethods (Xu et al., 2005, “Inkjet printing of viable mammalian cells,”Biomaterials, 26(1), pp. 93-99). Commercial inkjet cartridges (HP51626A)were emptied and rinsed thoroughly with distilled water and 70% ethanol.The entire printer assembly, including the cartridges, was kept in alaminar flow biosafety cabinet under ultraviolet light overnight priorto use. A pattern that consisted of rows of rectangles was designedusing the Microsoft Word software (Microsoft, Redmond, Wash.). Thealginate/cell print suspensions were loaded into the cartridges andprinted drop-by-drop into 35-mm petri dishes, which contained CaCl₂solutions at different ionic strengths (0.05M, 0.1M, 0.5M, and 1M). Amagnetic stir system was mounted under the inkjet printer and used toprevent aggregation of micro-particles during the printing process.Subsequently, the CaCl₂ solutions were removed from the Petri dishes andculture medium was carefully introduced. The samples were maintained atstandard culture conditions.

Using this modified HP DeskJet 550C printer, alginate microparticlescontaining one to several insulin producing cells (beta-TC6) werefabricated by printing a composition with the cells and sodium alginatesuspension into a CaCl₂ solution, generating microparticles of 30-60 μmin diameter at a rate as high as 55,000 particles per second. Cellsurvival assays showed that more than 89% of printed cells survived thefabrication process.

The effects of varying print conditions on cell distribution andmorphology of the printed microparticles were evaluated by changingsodium alginate concentrations, CaCl₂ concentrations, and/or cellconcentrations for printing. The efficiency of the fabrication methodwas evaluated by counting the total number of particles with respect totime.

The viability of the beta-TC6 cells contained in the printed alginatemicroparticles was evaluated by a two-color fluorescence live/dead assayusing a solution consisting of 2 μM calcein AM and 4 μM ethidiumhomodimer (EthD-1; Molecular Probes, OR) in 10 ml phosphate bufferedsaline (PBS). Printed samples with beta-TC6 cells were rinsed to removeresidual serum, and 2 ml of viability testing solution was added to eachsample. The samples were incubated for 30 min at 37° C. followed byrinse with PBS. The samples were viewed using a fluorescent microscopeand the viability of the cells was evaluated by counting the number ofcells stained with calcein AM (green), and this number was compared tothe total number of cells. The viability results of the printed sampleswere compared with the controls, in which beta-TC6 cells were manuallyplated onto a standard tissue culture plate (BD Biosciences, San Jose,Calif.).

The insulin secreted from the printed cell particles was analyzed withan ultrasensitive mouse insulin enzyme-linked immunosorbent assay(ELISA) kit (Mercodia, NC). The media was changed every two days andcollected 4 and 6 days after culture for analysis. As per themanufacturer's protocol, insulin levels were detected upon comparison ofthe unknown samples to the provided standards. The ELISA procedureconsisted of a 2 hour incubation of unknown and standard samples with anenzyme conjugated insulin-specific antibody, followed by 6 washes and afinal incubation with the soluble substrate 3-3′,5-5′tetramethylbenzidine (TMB) before reading at 450 nm.

All results were presented as mean±standard deviation. The grouped datawere statistically compared with Analysis of Variance (ANOVA) and atwo-sample Student's t-test.

The printed beta-TC6 cells demonstrated continuous insulin secretionover a 6-day period, which suggests that the printed cells are able tomaintain normal cellular function within the microparticles.

The printing conditions, such as cell number, alginate concentration,and ionic strengths of CaCl₂, influenced cellular distribution andgeometry of the printed particles. The droplets ejected from an inkjetprinter according to this Example range from 8 to 95 pL (Xu et al.,2004, “Construction of high-density bacterial colony arrays and patternsby the ink-jet method,” Biotechnol Bioeng, 85(1), pp. 29-33), whichpermit the generation of micro-scale particles that contain singlecells. Furthermore, the printing process according to this Exampleinvolves a high firing frequency (5-20 kHz) that can produce over250,000 droplets per second (Xu et al., 2006, “Viability andelectrophysiology of neural cell structures generated by the inkjetprinting method,” Biomaterials, 27(19), pp. 3580-3588), which issuitable for high throughput production of micro-particles.

The printing process readily produced microparticles that containedsingle or a few cells, indicating that printing techniques can be usedfor direct production of cell-containing microparticles. FIG. 11 showsthe morphology of the printed cell microparticles. Most particlescontained single to just a few cells, and only a small number ofparticles was found empty (FIG. 11).

The stability of the printed alginate particles was evaluated byculturing the printed microparticle samples over a period of 1 month.The microparticles maintained their structural integrity, and the cellsremained viable within each particle. The production rate was measuredby counting the total number of printed microparticles with respect totime. The calculations showed that the thermal inkjet printer was ableto generate cell-containing microparticles at a rate of approximately55,000 microparticles per second.

To determine the effects of cell concentration on the number of cellsencapsulated within the printed microparticles, print suspensions withdifferent beta-TC6 cell concentrations (2×10⁶, 6×10⁶, and 12×10⁶cells/ml) were tested. The number of cells within the printedmicroparticle increased as the cell concentration in the print solutionincreased. However, the number of microparticles containing only asingle cell decreased significantly with increase in cell concentration(FIG. 12) (p<0.01, n=10). The concentration of 6×10⁶ cells/ml resultedin one or more cells being present in about 50% of all particles. Ofthose cell-containing particles, about 70% contained a single cell.These findings indicate that an appropriate concentration of cells inthe printing solution would permit a high yield of microparticlescontaining single cells.

The effects of printing parameters on microparticle geometry were alsoinvestigated using different ionic strengths of CaCl₂ solutions.Although the printed particles had similar shapes in each CaCl₂solution, the diameter of the particles was dependent on the CaCl₂ ionicstrength. As shown in FIG. 13A, the printed particles had relativelylarger diameters when higher CaCl₂ ionic strength was used (p<0.01,n=10). In contrast, an increase in the alginate concentration did notchange the particle diameter significantly (FIG. 13B) (p>0.01, n=10).However, changes in the alginate concentration caused a marked variationin particle geometry (FIG. 13C). Most particles printed from 1% alginatewere found as round structures, while most particles generated from 0.5%alginate were shaped irregularly. Microparticles from 2% alginatedemonstrated long tailed shapes, suggesting that the viscosity of thealginate solution used may play a role in the final outcome of particlegeometry.

Survival rate of the printed beta-TC6 cells within the particles wasanalyzed by a commercial cell survival assay and compared with thecontrols (n=10), which were prepared by manually placing the cells ontostandard tissue culture plates. The cell/dead assay showed that morethan 89% of printed cells remained viable within the microparticles 1day after printing (FIG. 14). The printed control particles showedsimilar viability (93.1±4.6%) (P>0.05, n=10).

To determine whether normal cell function can be retained throughout theprinting process, microparticles containing cells were analyzed forinsulin production using an ultra-sensitive ELISA method. The insulinsecretion profiles for cultures of beta-TC6 cell-containingmicroparticles displayed a continuous insulin secretion pattern duringthe entire 6-day period of the study. The secreted insulinconcentrations from the printed particles were similar to non-printedcontrols, which were prepared by manually placing the cells ontostandard tissue culture plates. This indicates that normal cellularfunction can be preserved by the inkjet printing technique (FIG. 15).

Inkjet printing is considered to be one of the most promising newmethods for the selective and precise deposition of functional materialsto target locations (Park et al., 2006, “Control of colloidal particledeposit patterns within picoliter droplets ejected by ink-jet printing,”Langmuir, 22(8), pp. 3506-3513). The print heads of a thermal inkjetprinter are typically equipped with 30-200 μm capillary channels(Ringeisen et al., 2006, “Jet-based methods to print living cells,”Biotechnol J, 1(9), pp. 930-948) to allow for the delivery of individualdroplets with small volumes ranging from 8 to 95 pL per droplet (Xu etal., 2004, “Construction of high-density bacterial colony arrays andpatterns by the ink-jet method,” Biotechnol Bioeng, 85(1), pp. 29-33).This unique feature permits the inkjet printer to serve as amicro-fabrication tool for the generation of micro-scale particles.

We hypothesized that cells would become entrapped within the dropletsduring printing and produce single-cell containing microparticles ifboth droplets and cells were printed simultaneously. In this study wedemonstrate that insulin producing beta-TC6 cells can be combined withalginate solutions and printed into CaCl₂ solutions. We show thatprinted alginate microparticles are able to entrap a single to severalcells that can be maintained stably for up to one month post-printing.

The ability to control the size of the printed particles is critical forapplication in vivo. The diameter of individual mammalian cells in anunattached status generally ranges from 10-30 μm. Therefore, the size ofthe particles generated by the printing method in this study iscomparable to these cells, considering the actual size ranges that werefabricated in this study. However, the size of the particles can becontrolled by modifying the printing parameters such as varying theionic strengths of CaCl₂ solution. These diameter differences may be dueto the surface gelling mechanism, in which higher CaCl₂ ionic strengthsresult in immediate gelling at the surface with minimal penetration ofCa++ ions into the alginate droplets (Boland et al., 2006, “Applicationof inkjet printing to tissue engineering,” Biotechnol J, 1(9), pp.910-917). The reduced diameter of the microparticles, which approximatesthat of a single cell, may extend the viability and function of theentrapped cells within the microparticles.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1.-10. (canceled)
 11. A method of making a biodegradable scaffold havingcells seeded therein, comprising: (a) forming a biodegradable substrateby electrospinning; and then (b) printing viable cells on said substratewith an inkjet printing device, to make said biodegradable scaffold. 12.The method of claim 11, wherein the forming step and the printing stepare each performed two or more times in sequence to make a biodegradablescaffold having multiple layers.
 13. The method of claim 11, whereinsaid cells are provided in a liquid carrier.
 14. The method of claim 13,wherein said liquid carrier comprises collagen and/or elastin.
 15. Themethod of claim 13, wherein said liquid carrier comprises a hydrogel.16. The method of claim 13, wherein said liquid carrier comprises atleast one support compound.
 17. The method of claim 11, wherein saidsubstrate comprises a synthetic polymer selected from the groupconsisting of polycaprolactone (PCL), poly(_(D,L)-lactide-co-glycolide)(PLGA), polylactide (PLA), poly(lactide-co-caprolactone) (PLCL), andcombinations thereof.
 18. The method of claim 11, wherein said cells arestem cells.
 19. The method of claim 11, wherein said cells are selectedfrom the group consisting of cartilage cells, bone cells, muscle cellsand skin cells.
 20. The method of claim 11, wherein at least 50% of thecells by number are viable 24 hours after said printing step.
 21. Themethod of claim 11, wherein at least 75% of the cells by number areviable 24 hours after said printing step.
 22. The method of claim 11,wherein at least 90% of the cells by number are viable 24 hours aftersaid printing step.
 23. The method of claim 11, wherein said inkjetprinting device is selected from the group consisting of thermal inkjetprinters and piezoelectric inkjet printers.
 24. The method of claim 11,wherein said inkjet printing device comprises at least one inkjetcartridge, and wherein said inkjet cartridge is selected from the groupconsisting of thermal inkjet printer cartridges and piezoelectric inkjetprinter cartridges.
 25. The method of claim 11, further comprising thestep of loading a composition to be printed into said printer cartridgeprior to the step of printing, said composition comprising cells and aliquid carrier.
 26. The method of claim 11, wherein said forming stepand said printing step are carried out by electrospinning and printinginto a media.
 27. A biocompatible scaffold produced by the method ofclaim
 12. 28. A method of treating a subject in need thereof comprisingimplanting the biodegradable scaffold claim 27 into said subject. 29.The method of claim 28, wherein said cells are selected from the groupconsisting of cartilage cells, bone cells, muscle cells and skin cells30-50. (canceled)